The present invention relates generally to genetic sequences encoding flavonoid pathway metabolising enzymes and more particularly to flavonoid 3xe2x80x2-hydroxylase (hereinafter referred to as xe2x80x9cF3xe2x80x2Hxe2x80x9d) or derivatives thereof and their use in the manipulation of pigmentation in plants and other organisms.
Bibliographic details of the publications referred to by the author in this specification are collected at the end of the description. Sequence Identity Numbers (SEQ ID NOs) for the nucleotide and amino acid sequences referred to in the specification and claims are defined following the bibliography. A summary of the SEQ ID NOs, and the sequences to which they relate, is provided prior to the Examples.
Throughout this specification, unless the context requires otherwise, the word xe2x80x9ccomprisexe2x80x9d, or variations such as xe2x80x9ccomprisesxe2x80x9d or xe2x80x9ccomprisingxe2x80x9d, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
The rapidly developing sophistication of recombinant DNA technology is greatly facilitating research and development in a range of biotechnology related industries. The horticultural industry has become a recent beneficiary of this technology which has contributed to developments in disease resistance in plants and flowers exhibiting delayed senescence after cutting. Some attention has also been directed to manipulating flower colour.
The flower industry strives to develop new and different varieties of flowering plants. An effective way to create such novel varieties is through the manipulation of flower colour. Classical breeding techniques have been used with some success to produce a wide range of colours for most of the commercial varieties of flowers. This approach has been limited, however, by the constraints of a particular species"" gene pool and for this reason it is rare for a single species to have a full spectrum of coloured varieties. In addition, traditional breeding techniques lack precision. The aesthetic appeal of the flower is a combination of many factors such as form, scent and colour; modification of one character through hybridization can often be at the expense of an equally valuable feature. The ability to genetically engineer precise colour changes in cutflower and ornamental species would offer significant commercial opportunities in an industry which has rapid product turnover and: where novelty is an important market characteristic.
Flower colour is predominantly due to two types of pigment: flavonoids and carotenoids. Flavonoids contribute to a range of colours from yellow to red to blue. Carotenoids impart an orange or yellow tinge and are commonly the major pigment in yellow or orange flowers. The flavonoid molecules which make the major contribution to flower colour are the anthocyanins which are glycosylated derivatives of cyanidin, delphinidin, petunidin, peonidin, malvidin and pelargonidin, and are localised in the vacuole. The different anthocyanins can produce marked differences in colour. Flower colour is also influenced by co-pigmentation with colourless flavonoids, metal complexation, glycosylation, acylation and vacuolar pH (Forkmann, 1991).
The biosynthetic pathway for the flavonoid pigments (hereinafter referred to as the xe2x80x9cflavonoid pathwayxe2x80x9d) is well established and is shown in FIGS. 1a and 1b (Ebel and Hahlbrock, 1988; Hahlbrock and Grisebach, 1979; Wiering and De Vlaming, 1984; Schram et al., 1984; Stafford, 1990; Van Tunen and Mol, 1990; Dooner et al, 1991; Martin and Gerats, 1993; Holton and Cornish, 1995). The first committed step in the pathway involves the condensation of three molecules of malonyl-CoA with one molecule of peoumaroyl-CoA. This reaction is catalysed by the enzyme chalcone synthase (CHS). The product of this reaction, 2xe2x80x2,4,4xe2x80x2,6xe2x80x2, tetrahydroxy-chalcone, is normally rapidly isomerized to produce naringenin by the enzyme chalcone flavanone isomerase (CHI). Naringenin is subsequently hydroxylate at the 3 position of the central ring by flavanone 3-hydroxylase (F3H) to produce dihydrokaempferol (DHK).
The pattern of hydroxylation of the B-ring of DHK plays a key role in determining petal colour. The B-ring can be hydroxylated at either the 3xe2x80x2, or both the 3xe2x80x2 and 5xe2x80x2 positions, to produce dihydroquercetin (DHQ) and dihydromyricetin (DHM), respectively. Two key enzymes involved in this pathway are flavonoid 3xe2x80x2-hydroxylase and flavonoid 3xe2x80x2,5xe2x80x2-hydroxylase, both of the cytoclrome P450 class. Cytochrome P450 enzymes are widespread in nature and genes have been isolated and sequenced from vertebrates, insects, yeasts, fungi, bacteria and plants.
Flavonoid 3xe2x80x2-hydroxylase acts on DHK to produce DHQ and on naringenin to produce eriodictyol. Reduction and glycosylation of DHQ produces the cyanidin-glycoside and peonidin-glycoside pigments which, in many plant species (for example rose, carnation and chrysanthemum), contribute to red and pink flower colour. The synthesis of these anthocanins can also result in other flower colours. For example, blue cornflowers contain emcyan. The ability to control flavonoid 3xe2x80x2-hydroxylase activity, or other enzymes involved in the flavonoid pathway, in flowering plants would provide a means to manipulate petal colour. Different coloured versions of a single cultivar could thereby be generated and in some instances a single species would be able to produce a broader spectrum of colours.
A nucleotide sequence (referred to herein as SEQ ID NO:26) encoding a petunia flavonoid 3xe2x80x2-hydroxylase has been cloned (see International Patent Application No. PCT/AU93/00127 [WO 93/120206]). However, this sequence was inefficient in its ability to modulate the production of 3xe2x80x2-hydroxylated anhocyanins in plants There is a need, therefore, to identify further genetic sequences encoding flavonoid 3xe2x80x2-hydroxylases which efficiently modulate the hydroxylation of flavonoid compounds in plants. More particularly, there is a need to identify further genetic sequences encoding flavonoid 3xe2x80x2-hydroxylases which efficiently modulate the production of 3xe2x80x2-hydroxylated anthocyanins in plants.
In accordance with the present invention, genetic sequences encoding flavonoid 3xe2x80x2-hydroxylase have been identified and cloned. The recombinant genetic sequences of the present invention permit the modulation of expression of genes encoding this enzyme by, for example, de novo expression, over-expression, suppression, antisense inhibition and ribozyme activity. The ability to control flavonoid 3xe2x80x2-hydroxylase synthesis in plants permits modulation of the composition of individual anthocyanins as well as alteration of relative levels of flavonols and anthocyanins, thereby enabling the manipulation of tissue colour, such as petals, leaves, seeds and fruit. The present invention is hereinafter described in relation to the manipulation of flower colour but this is done with the understanding that it extends to manipulation of other plant tissues, such as leaves, seeds and fruit.
Accordingly, one aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding a flavonoid 3xe2x80x2-hydroxylase or a derivative thereof wherein said flavonoid 3xe2x80x2-hydroxylase or its derivative is capable of more efficient modulation of hydroxylation of flavonoid compounds in plants than is a flavonoid 3xe2x80x2-hydroxylase encoded by the nucleotide sequence set forth in SEQ ID NO:26.
Efficiency as used herein relates to the capability of the flavonoid 3xe2x80x2-hydroxylase enzyme to hydroxylate flavonoid compounds in a plant cell. This provides the plant with additional substrates for other enzymes of the flavonoid pathway able to further modify this molecule, via, for example, glycosylation, acylation and rhamnosylation, to produce various anthocyanins which contribute to the production of a range of colours. The modulation of 3xe2x80x2-hydroxylated anthocyanins is thereby permitted. Efficiency is conveniently assessed by one or more parameters selected from: extent of transcription, as determined by the amount of mRNA produced; extend of hydroxylation of naringenin and/or DHK; extent of translation of mRNA, as determined by the amount of translation product produced; extent of production of anthocyanin derivatives of DHQ or DHM; the extent of effect on tissue colour, such as flowers, seeds, leaves or fruits.
Another aspect of the present invention is directed to an isolated nucleic acid molecule comprising a sequence of nucleotides which maps to the genetic locus designated Ht1 or Ht2 in petunia, or to equivalent such loci in other flowering plant species, and wherein said isolated nucleic acid molecule encodes, or is complementary to a sequence which encodes, a flavonoid 3xe2x80x2-hydroxylase.
A further aspect of the present invention contemplates an isolated nucleic acid molecule comprising a sequence of nucleotides which corresponds to the genetic locus designated Ht1 or Ht2 in petunia, or to loci in other flowering plant species which contain sequences which control production of 3xe2x80x2-hydroxylated flavonoids, and wherein said isolated nucleic acid molecule encodes a flavonoid 3xe2x80x2-hydroxylase or a derivative thereof which is capable of more efficient conversion of DHK to DHQ in plants than is the flavonoid 3xe2x80x2-hydroxylase set forth in SEQ ID NO:26.
In accordance with the above aspects of the present invention there is provided a nucleic acid molecule comprising a nucleotide sequence or complementary nucleotide sequence substantially as set forth in SEQ ID NO:1 or having at least about 60% similarity thereto or capable of hybridising to the sequence set forth in SEQ ID NO:1 under low stringency conditions.
In a related embodiment, there is provided a nucleic acid molecule comprising a nucleotide sequence or complementary nucleotide sequence substantially as set forth in SEQ ID NO:3 or having at least about 60% similarity thereto or capable of hybridising to the sequence set forth in SEQ ID NO:3 under low stringency conditions.
In another related embodiment, the present invention is directed to a nucleic acid molecule comprising a nucleotide sequence or complementary nucleotide sequence substantially as set forth in SEQ ID NO:5 or having at least about 60% similarity thereto or capable of hybridising to the sequence set forth in SEQ ID, NO:5 under low stringency conditions.
Yet another related embodiment provides a nucleic acid molecule comprising a nucleotide sequence or complementary nucleotide sequence substantially as set forth in SEQ ID NO:7 or having at least about 60% similarity thereto or capable of hybridising to the sequence set forth in SEQ ID NO:7 under low stringency conditions.
Still yet a further embodiment of the present invention relates to a nucleic acid molecule comprising a nucleotide sequence or complementary nucleotide sequence substantially as set forth in SEQ ID NO:9 or having at least about 60% similarity to the coding region thereof or capable of hybridising to the sequence set forth in SEQ ID NO:9 under low stringency conditions.
In another further embodiment, there is provided a nucleic acid molecule comprising a nucleotide sequence or complementary nucleotide sequence substantially as set forth in SEQ ID NO:14 or having at least about 60% similarity thereto or capable of hybridising to the sequence set forth in SEQ ID NO:14 under low stringency conditions.
In yet another further embodiment, the present invention is directed to a nucleic acid molecule comprising a nucleotide sequence or complementary nucleotide sequence substantially as set forth in SEQ ID NO:16 or having at least about 60% similarity thereto or capable of hybridising to the sequence set forth in SEQ ID NO:16 under low stringency conditions.
Still yet another further embodiment provides a nucleic acid molecule comprising a nucleotide sequence or complementary nucleotide sequence substantially as set forth in SEQ ID NO:18 or having at least about 60% similarity thereto or capable of hybridising to the sequence set forth in SEQ ID NO:18 under low stringency conditions.
Moreover, yet a further embodiment of the present invention relates to a nucleic acid molecule comprising a nucleotide sequence or complementary nucleotide sequence substantially as set forth in SEQ ID NO:20 or having at least about 60% similarity thereto or capable of hybridising to the sequence set forth in SEQ ID NO:20 under low stringency conditions.
Still yet another further embodiment is directed to a nucleic acid molecule comprising a nucleotide sequence or complementary nucleotide sequence substantially as set forth in SEQ ID NO:22 or having at least about 60% similarity thereto or capable of hybridising to the sequence set forth in SEQ ID NO:22 under low stringency conditions.
In still yet another further embodiment, the present invention provides a nucleic acid molecule comprising a nucleotide sequence or complementary nucleotide sequence substantially as set forth in SEQ ID NO:24 or having at least about 60% similarity thereto or capable of hybridising to the sequence set forth in SEQ ID NO:24 under low stringency conditions.
In a particularly preferred embodiment there is provided an isolated nucleic acid molecule comprising a nucleotide sequence or complementary nucleotide sequence substantially as set forth in SEQ ID NO:1 or having at least about 60% similarity thereto or capable of hybridising to the sequence set forth in SEQ ID NO:1 under low stringency conditions, wherein said nucleotide sequence maps to the genetic locus designated Ht1 or Ht2 in petunia, or to equivalent such loci in other flowering plant species, and wherein said isolated nucleic acid molecule encodes, or is complementary to a sequence which encodes, a flavonoid 3xe2x80x2-hydroxylase.
Reference herein to a low stringency at 42xc2x0 C. includes and encompasses from at least about 1% to at least about 15% formamide and from at least about 1M to at least about 2M salt for hybridization, and at least about 1M to at least about 2M salt for washing conditions. Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% to at least about 30% formamide and from at least about 0.5M to at least about 0.9M salt for hybridization, and at least about 0.5M to at least about 0.9M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% to at least about 50% formamide and from at least about 0.01M to at least about 0.15M salt for hybridization, and at least about 0.01M to at least about 0.15M salt for washing conditions. Hybridization may be carried out at different temperatures and, where this occurs, other conditions may be adjusted accordingly.
Another aspect of the present invention provides a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding an amino acid sequence substantially as set forth in SEQ ID NO:2 or an amino acid sequence, having at least about 50% similarity thereto.
In a related embodiment, there is provided a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding an amino acid sequence substantially as set forth in SEQ ID NO:4 or an amino acid sequence having at least about 50% similarity thereto.
A further related embodiment of the present invention is directed to a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary toga sequence encoding an amino acid sequence substantially as set forth in SEQ ID NO:6 or an amino acid sequence having at least about 50% similarity thereto.
Still another related embodiment provides a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding an amino acid sequence substantially as set forth in SEQ ID NO:8 or an amino acid sequence having at least about 50% similarity thereto.
Yet still another related embodiment relates to a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding an amino acid sequence substantially as set forth in SEQ ID NO:10 or SEQ ID NO:11 or SEQ ID NO:12 or SEQ ID NO:13 or an amino acid sequence having at least about 50% similarity thereto.
In another further embodiment, there is provided a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding an amino acid sequence substantially as set forth in SEQ ID NO:15 or an amino acid sequence having at least about 50% similarity thereto.
In yet another further embodiment, the present invention is directed to a nucleic acid molecule-comprising a sequence of nucleotides encoding or complementary to a sequence encoding an amino acid sequence substantially as set forth in SEQ ID NO:17 or an amino acid sequence having at least about 50% similarity thereto.
Still yet another further embodiment provides a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding an amino acid sequence substantially as set forth in SEQ ID NO:19 or an amino acid sequence having at least about 50% similarity thereto.
Moreover, yet a further embodiment of the present invention relates to:: a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding an amino acid sequence substantially as set forth in SEQ ID NO:21 or an amino acid sequence having at least about 50% similarity thereto.
Still yet another further embodiment is directed to a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding an amino acid sequence substantially as set forth in SEQ ID NO:23 or an amino acid sequence having at least about 50% similarity thereto.
In still yet another further embodiment, the present invention provides a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding an amino acid sequence substantially as set forth in SEQ ID NO:25 or an amino acid sequence having at least about 50% similarity thereto.
In a particularly preferred embodiment there is provided an isolated nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding an amino acid sequence substantially as set forth in SEQ ID NO:2 or an amino acid sequence having at least about 50% similarity thereto, wherein said sequence of nucleotides maps to the genetic locus designated Ht1 or Ht2 in petunia, or to equivalent such loci in other flowering plant species, and wherein said isolated nucleic acid molecule encodes, or is complementary to a sequence which encodes, a flavonoid 3xe2x80x2-hydroxylase or a derivative therof.
The term xe2x80x9csimilarityxe2x80x9d as used herein includes exact identity between compared sequences, at the nucleotide or amino acid level. Where there is non-identity at the nucleotide level, xe2x80x9csimilarityxe2x80x9d includes differences between sequences which result in different amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. Where there is non-identity at the amino acid level, xe2x80x9csimilarityxe2x80x9d includes amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels.
The nucleic acid molecule defined by SEQ ID NO:1 encodes a flavonoid 3xe2x80x2-hydroxylase from petunia. Examples of other suitable F3xe2x80x2H genes are from carnation (SEQ ID NO:3), snapdragon (SEQ ID NO:5), arabidopsis (SEQ ID NO:7), arabidopsis genomic DNA clone (SEQ ID NO:9), rose (SEQ ID NO:14), chrysanthemum (SEQ ID NO:16), torenia (SEQ ID NO:18), Japanese morning glory (SEQ ID NO:20), gentian (SEQ ID NO:22) and lisianthus (SEQ ID NO:24). Although the present invention is particularly exemplified by the aforementioned F3xe2x80x2H genes, the subject invention extends to F3xe2x80x2H genes from any species of plant provided that the F3xe2x80x2H gene has at least about 60% similarity at the nucleotide level to a nucleic acid molecule selected from SEQ ID NO:1 or 3 or 5 or 7 or 14 or 16 or 18 or 20 or 22 or 24, or at least about 50% similarity at the amino acid level to an amino acid molecule selected from SEQ ID NO:2 or 4 or 6 or 8 or 10, 11, 12, 13 or 15 or 17 or 19 or 21 or 23 or 25. The subject invention further extends to F3xe2x80x2H genes from any species of plant provided that the F3xe2x80x2H gene has at least about 60% similarity at the nucleotide level to the coding region of SEQ ID NO:9.
The nucleic acid molecules of the present invention are generally genetic sequences in a non-naturally-occurring condition. Generally, this means isolated away from its natural state or synthesized or derived in a non-naturally-occurring environment. More specifically, it includes nucleic acid molecules formed or maintained in vitro, including genomic DNA fragments, recombinant or synthetic molecules and nucleic acids in combination with heterologous nucleic acids. It also extends to the genomic DNA or cDNA or part thereof encoding F3xe2x80x2H or part thereof in reverse orientation relative to its or another promoter. It further extends to naturally-occurring sequences following at least a partial purification relative to other nucleic acid sequences.
The term xe2x80x9cnucleic acid moleculexe2x80x9d includes a nucleic acid isolate and a genetic sequence and is used herein in its most general sense and encompasses any contiguous series of nucleotide bases specifying directly, or via a complementary series of bases, a sequence of amino acids in a F3xe2x80x2H. Such a sequence of amino acids may constitute a full-length F3xe2x80x2H or an active truncated form thereof or may correspond to a particular region such as an N-terminal, C-terminal or internal portion of the enzyme. The nucleic acid molecules contemplated herein also encompass oligonucleotides useful as genetic probes or as xe2x80x9cantisensexe2x80x9d molecules capable of regulating expression of the corresponding gene in a plant. An xe2x80x9cantisense moleculexe2x80x9d as used herein may also encompass a gene construct comprising the structural genomic or cDNA gene or part thereof in reverse orientation relative to its own or another promoter. Accordingly, the nucleic acid molecules of the present invention may be suitable for use as cosuppression molecules, ribozyme molecules, sense molecules and antisense molecules to modulate levels of 3xe2x80x2-hydroxylated anthocyanins.
In one embodiment, the nucleic acid molecule encoding F3xe2x80x2H or various derivatives thereof is used to reduce the activity of an endogenous F3xe2x80x2H, or alternatively the nucleic acid molecule encoding this enzyme or various derivatives thereof is used in the antisense orientation to reduce activity of the F3xe2x80x2H. Although not wishing to limit the present invention to any one theory, it is possible that the introduction of the nucleic acid molecule into a cell results in this outcome either by decreasing transcription of the homologous endogenous gene or by increasing turnover of the corresponding mRNA. This may be achieved using gene constructs containing F3xe2x80x2H nucleic acid molecules or various derivatives thereof in either the sense or the antisense orientation. In a further alternative, ribozymes could be used to inactivate target nucleic acid molecules. Alternatively, the nucleic acid molecule encodes a functional F3xe2x80x2H and this is used to elevate levels of this enzyme in plants.
Reference herein to the altering of flavonoid F3xe2x80x2H activity relates to an elevation or reduction in activity of up to 30% or more preferably of 30-50%, or even more preferably 50-75% or still more preferably 75% or greater above or below the normal endogenous or existing levels of activity. The level of activity can be readily assayed using a modified version of the method described by Stotz and Forkmann (1982) (see Example 7) or by assaying for the amount of F3xe2x80x2H product such as quercetin, cyanidin or peonidin as set forth in Example 5.
The present invention further extends to nucleic acid molecules in the form of oligonucleotide primers or probes capable of hybridizing to a portion of the nucleic acid molecules contemplated above, and in particular those selected from the nucleic acid molecules set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 14, 16, 18, 20, 22 or 24 under high, preferably under medium and most preferably under low stringency conditions. Preferably the portion corresponds to the 5xe2x80x2 or the 3xe2x80x2 end of the F3xe2x80x2H gene. For convenience the 5xe2x80x2 end is considered herein to define a region substantially between the 5xe2x80x2 end of the primary transcript to a centre portion of the gene, and the 3xe2x80x2 end is considered herein to define a region substantially between the centre portion of the gene and the 3xe2x80x2 end of the primary transcript. It is clear, therefore, that oligonucleotides or probes may hybridize to the 5xe2x80x2 end or the 3xe2x80x2 end or to a region common to both the 5xe2x80x2 and the 3xe2x80x2 ends.
The nucleic acid molecule or its complementary form may encode the fill-length enzyme or a part or derivative thereof. By xe2x80x9cderivativexe2x80x9d is meant any single or multiple amino acid substitutions, deletions, and/or additions relative to the naturally-occurring enzyme and includes parts, fragments, portions, fusion molecules, homologues and analogues. In this regard, the nucleic acid includes the naturally-occurring nucleotide sequence encoding F3xe2x80x2H or may contain single or multiple nucleotide substitutions, deletions and/or additions to said naturally-occurring sequence. A fusion molecule may be a fusion between nucleotide sequences encoding two or more F3xe2x80x2Hs, or a fusion between a nucleotide sequence encoding an F3xe2x80x2H and a nucleotide sequence encoding any other proteinaceous molecule. Fusion molecules are useful in altering substrate specificity.
A derivative of the nucleic acid molecule or its complementary form, or of a F3xe2x80x2H, of the present invention may also include a xe2x80x9cpartxe2x80x9d, whether active or inactive. An active or functional nucleic acid molecule is one which encodes an enzyme with F3xe2x80x2H activity. An active or functional molecule further encompasses a partially-active molecule; for example, an F3xe2x80x2H with reduced substrate specificity would be regarded as partially active A derivative of a nucleic acid molecule may be useful as an oligonucleotide probe, as a primer for polymerase chain reactions or in various mutagenic techniques, for the generation of antisense molecules or in the construction of ribozymes. They may also be useful in developing co-suppression constructs. The nucleic acid molecule according to this aspect of the present invention may or may not encode a functional F3xe2x80x2H. A xe2x80x9cpartxe2x80x9d may be derived from the 5xe2x80x2 end or the 3xe2x80x2 end or a region common to both the 5xe2x80x2 and the 3xe2x80x2 ends of the nucleic acid molecule.
Amino acid insertional derivatives of the F3xe2x80x2H of the present invention include amino and/or carboxyl terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterised by the removal of one or more amino acids from the sequence Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. Typical substitutions are those made in accordance with Table 1 below.
Where the F3xe2x80x2H is derivatised by amino acid substitution, the amino acids are generally replaced by other amino acids having lie properties, such as hydrophobicity, hydrophilicity, electronegativity, bulky side chains and the like. Amino acid substitutions are typically of single residues. Amino acid insertions will usually be in the order of about 1-10 amino acid residues and deletions will range from about 1-20 residues. Preferably, deletions or insertions are made in adjacent pairs, i.e. a deletion of two residues or insertion of two residues.
The amino acid variants referred to above may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis (Merrifield, 1964) and the like, or by recombinant DNA manipulations. Techniques for making substitution mutations at predetermined sites in DNA having known or partially known sequence are well known and include, for example, M13 mutagenesis. The manipulation of DNA sequence to produce variant proteins which manifest as substitutional, insertional or deletional variants are conveniently described, for example, in Sambrook et al. (1989).
Other examples of recombinant or synthetic mutants and derivatives of the F3xe2x80x2H of the present invention include single or multiple substitutions, deletions and/or additions of any molecule associated with the enzyme such as carbohydrates, lipids and/or proteins or polypeptides.
The terms xe2x80x9canaloguesxe2x80x9d and xe2x80x9cderivativesxe2x80x9d also extend to any chemical equivalents of the F3xe2x80x2H, whether functional or not, and also to any amino acid derivative described above. Where the xe2x80x9canaloguesxe2x80x9d and xe2x80x9cderivativesxe2x80x9d of this aspect of the present invention are non-functional, they may act as agonists or antagonists of F3xe2x80x2H activity. For convenience, reference to xe2x80x9cF3xe2x80x2Hxe2x80x9d herein includes reference to any derivatives, including parts, mutants, fragments, homologues or analogues thereof.
The present invention is exemplified using nucleic acid sequences derived from petunia, carnation, rose, snapdragon, arabidopsis, chrysanthemum, lisianthus, torenia, morning glory and gentian, since these represent the most convenient and preferred sources of material to date. However, one skill(A in the art will immediately appreciate that similar: sequences can be isolated from any number of sources such as other plants or certain microorganisms. Examples of other plants include, but are not limited to, maize, tobacco, cornflower, pelargonium, apple, gerlera and african violet. All such nucleic acid sequences encoding directly or indirectly a flavonoid pathway enzyme and in particular F3xe2x80x2H, regardless of their source, are encompassed by the present invention.
The nucleic acid molecules contemplated herein may exist in either orientation alone or in combination with a vector molecule, for example an expression-vector. The term vector molecule is used in its broadest sense to include any intermediate vehicle for the nucleic acid molecule, capable of facilitating transfer of the nucleic acid into the plant cell and/or facilitating integration into the plant genome. An intermediate vehicle may, for example, be adapted for use in electroporation, microprojectile bombardment, Agrobacterium-mediated transfer or insertion via DNA or RNA viruses. The intermediate vehicle and/or the nucleic acid molecule contained therein may or may not need to be stably integrated into the plant genome. Such vector molecules may also replicate and/or express in prokaryotic cells. Preferably, the vector molecules or parts thereof are capable of integration into the plant genome. The nucleic acid molecule may additionally contain a promoter sequence capable of directing expression of the nucleic acid molecule in a plant cell. The nucleic acid molecule and promoter may also be introduced into the cell by any number of means such as those described above.
In accordance with the present invention, a nucleic acid molecule encoding a F3xe2x80x2H or a derivative or part thereof may be introduced into a plant in either orientation to allow, permit or otherwise facilitate manipulation of levels of production of mRNA in the cytoplasm and/or production of enzyme from the mRNA, thereby providing a means either to convert DHK and/or other suitable substrates, if synthesised in the plant cell, ultimately into anthocyanin derivatives of anthocyanidins such as cyanidin and/or peonidin, or alternatively to inhibit such conversion of metabolites by reducing or eliminating endogenous or existing F3xe2x80x2H activity. The production of mRNA in the cytoplasm and/or production of enzyme from the mRNA, is referred to herein as xe2x80x9cexpressionxe2x80x9d. The production of anthocyanins contributes to the production of a red or blue flower colour. Expression of the nucleic acid molecule in either orientation in the plant may be constitutive, inducible or developmental, and may also be tissue-specific.
According to this aspect of the present invention there is provided a method for producing a transgenic plant capable of synthesizing F3xe2x80x2H or functional derivatives thereof, said method comprising stably transforming a cell of a suitable plant with a nucleic acid molecule which comprises a sequence of nucleotides encoding said F3xe2x80x2H, under conditions permitting the eventual expression of said nucleic acid molecule, regenerating a transgenic plant from the cell and growing said transgenic plant for a time and under conditions sufficient to permit the expression of the nucleic acid molecule. The transgenic plant may thereby produce elevated levels of F3xe2x80x2H activity relative to the amount expressed in a comparable non-transgenic plant.
Another aspect of the present invention contemplates a method for producing a transgenic plant with reduced endogenous or existing F3xe2x80x2H activity, said method comprising stably transforming a cell of a suitable plant with a nucleic acid molecule which comprises a sequence of nucleotides encoding or complementary to a sequence encoding F3xe2x80x2H, regenerating a transgenic plant from the cell and where necessary growing said transgenic plant under conditions sufficient to permit the expression of the nucleic acid molecule.
Yet another aspect of the present invention contemplates a method for producing a genetically modified plant with reduced endogenous or existing F3xe2x80x2H activity, said method comprising altering the F3xe2x80x2H gene through modification of the endogenous sequences via homologous recombination from an appropriately altered F3xe2x80x2H gene or derivative or part thereof introduced into the plant cell, and regenerating the genetically modified plant from the cell.
In accordance with these aspects of the present invention the preferred nucleic acid molecules are substantially as set forth in SEQ ID NO:1, 3, 5, 7, 14. 16, 18, 20, 22, 24 or the coding region of 9, or have at least about 60% similarity thereto, or are capable of hybridising thereto under low stringency conditions.
In a preferred embodiment, the present invention contemplates a method for producing a transgenic flowering plant exhibiting altered flower colour, said method comprising stably transforming a cell of a suitable plant with a nucleic acid molecule of the present invention, regenerating a transgenic plant from the cell and growing said transgenic plant for a time and under conditions sufficient to permit the expression of the nucleic acid molecule into the F3xe2x80x2H enzyme. Alternatively, said method may comprise stably transforming a cell of a suitable plant with a nucleic acid molecule of the present invention or its complementary sequence, regenerating a transgenic plant from the cell and growing said transgenic plant for a time and under conditions sufficient to alter the level of activity of the endogenous or existing F3xe2x80x2H. Preferably, the altered level would be less than the endogenous or existing level of F3xe2x80x2H activity in a comparable non-transgenic plant.
In a related embodiment, the present invention contemplates a method for producing a flowering plant exhibiting altered flower colour, said method comprising alteration of the F3xe2x80x2H gene through modification of the endogenous sequences via homologous recombination from an appropriately altered F3xe2x80x2H gene or derivative thereof introduced into the plant cell and regenerating the genetically modified plant from the cell.
The nucleic acid molecules of the present invention may or may not be developmentally regulated. Preferably, the modulation of levels of 3xe2x80x2-hydroxylated anthocyanins leads to altered flower colour which includes the production of red flowers or other colour shades depending on the physiological conditions of the recipient plant. By xe2x80x9crecipient plantxe2x80x9d is meant a plant capable of producing a substrate for the F3xe2x80x2H enzyme, or producing the F3xe2x80x2H enzyme itself, and possessing the appropriate physiological properties and genotype required for the development of the colour desired. This may include but is not limited to petunia, carnation, chrysanthemum, rose, snapdragon, tobacco, cornflower, pelargonium, lisianthus, gerbera, apple, iris, lily, african violet, gentian, torenia and Japanese morning glory.
Accordingly, the present invention extends to a method for producing a transgenic plant capable of modulating levels of 3xe2x80x2-hydroxylated anthocyanins, said method comprising stably transforming a cell or group of cells of a suitable plant with a nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to a sequence encoding, F3xe2x80x2H or a derivative thereof, and regenerating a transgenic plant from said cell or cells.
One skilled in the art will immediately recognise the variations applicable to the methods of the present invention, such as increasing or decreasing the level of enzyme activity of the enzyme naturally present in a target plant leading to differing shades of colours.
The present invention, therefore, extends to all transgenic plants containing all or part of the nucleic acid module of the present invention and/or any homologues or related forms thereof or antisense forms of any of these and in particular those transgenic plants which exhibit altered flower colour. The transgenic plants may contain an introduced nucleic acid molecule comprising a nucleotide sequence encoding or complementary to a sequence encoding F3xe2x80x2H. Generally, the nucleic acid would be stably introduced into the plant genome, although the present invention also extends to the introduction of the F3xe2x80x2H nucleotide sequence within an autonomously-replicating nucleic acid sequence such as a DNA or RNA virus capable of replicating within the plant cell. The invention also extends to seeds from such transgenic plants. Such seeds, especially if coloured, will be useful as proprietary tags for plants.
A further aspect of the present invention is directed to recombinant forms of F3xe2x80x2H. The recombinant forms of the enzymes will provide a source of material for research to develop, for example, more active enzymes and may be useful in developing in vitro systems for production of coloured compounds.
Still a further aspect of the present invention contemplates the use of the genetic sequences described herein in the manufacture of a genetic construct capable of use in modulating levels of 3xe2x80x2-hydroxylated anthocyanins in a plant or cells of a plant.
Yet a further aspect of the present invention provides flowers and in particular cut flowers, from the transgenic plants herein described, exhibiting altered flower colour.
Another aspect of the present invention is directed to a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding, a F3xe2x80x2H or a derivative thereof wherein said nucleic acid molecule is capable of being expressed in a plant cell. The term xe2x80x9cexpressedxe2x80x9d is equivalent to the term xe2x80x9cexpressionxe2x80x9d as defined above.
The nucleic acid molecules according to this and other aspects of the invention allow, permit or otherwise facilitate increased efficiency in modulation of 3xe2x80x2-hydroxylated anthocyanins relative to the efficency of the pCGP619 cDNA insert contained in plasmid pCGP809, disclosed in International Patent Application No. PCT/AU93/00127 [WO 93/20206]. The term xe2x80x9cplant cellxe2x80x9d includes a single plant cell or a group of plant cells such as in a callus, plantlet or plant or parts thereof including flowers and seeds.
Another aspect of the present invention provides a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence of nucleotides encoding a F3xe2x80x2H, wherein the translation of the said nucleic acid molecule comprises the amino acid sequence RPPNSGA (SEQ ID NO:43). Preferably, the translation of the said nucleic acid molecule comprises the amino acid sequence RPPNSGAXHXAYNYXDL (SEQ ID NO:44) and still more preferably the translation of the said nucleic acid molecule comprises the amino acid sequence RPPNSGAXHXAYNYXDL[X]nGGEK (SEQ ID NO:45), where X represents any amino acid and [X]n represents an amino acid sequence of from 0 to 500 amino acids.
The present invention is further described by reference to the following non-limiting Figures and Examples.